Update on gene fusions and the emerging clinicopathological landscape of peritoneal and pleural mesotheliomas and other neoplasms

Background Mesothelioma is a rare and aggressive malignant neoplasm arising from mesothelial cells, which occasionally manifests recurrent fusions. EWSR1/FUS-CREB, YY1, MAP3K8, NR4A3, and ALK-rearranged proliferations have been reported in limited series with no clear histological or clinical correlations, limiting clinicians’ ability to assess prognosis and integrate these new entities into therapeutic decisions. The aim of this study was to better characterize these rearranged proliferations histologically, molecularly, and clinically. Methods Clinical, pathological, and comprehensive transcriptome and mutation data were collected for each case. Results A total of 41 tumors were included, encompassing 7 ALK, 10 MAP3K8, 4 NR4A3, 8 ESWR1/FUS::ATF1, 8 EWSR1::YY1, and 4 SUFU-fused cases. We found a female predominance, except for cases harboring NR4A3 and SUFU; and most patients were around 60 years of age, but those harboring ALK or EWSR1/FUS::ATF1 gene fusions were younger. Each group exhibited distinct histological, immunohistochemical, molecular features, and oncological courses. Specifically, MAP3K8 and ALK presented PAX8+ papillary proliferations, ESWR1/FUS::ATF1 and EWSR1::YY1 displayed angiomatoid fibrous histiocytoma-like patterns, while SUFU showcased ‘tissue culture’-like spindle cell proliferation. Poor prognosis factors were the pleural site, male sex, Ki67 ≥10%, and ESWR1/FUS::ATF1 or SUFU gene fusions. Conclusions This study significantly broadens the spectrum of mesothelial tumors associated with fusions, offering insight into novel epithelioid (mesothelial) proliferations with distinctive histological appearances, molecular profiles, and prognoses to guide adapted treatments for patients.


INTRODUCTION
Mesotheliomas are uncommon malignant tumors originating from mesothelial cells that line various serous cavities, including pleura, pericardium, peritoneum, and tunica vaginalis testis. 1,2These tumors are categorized into three primary histological types: epithelioid (EM), sarcomatoid (SM), and biphasic types, the epithelioid type representing in the pleura nearly 75% of the cases, the biphasic type 15% and the sarcomatoid type up to 10%. 3 Over the last decade, significant strides have been made in understanding mesothelioma pathogenesis.This progress has been driven by the integration of cutting-edge molecular and genomic techniques into research endeavors.5][6] Recently, small series of mesothelioma, mostly from the peritoneum, were reported to be driven by recurrent gene fusions, such as EWSR1/FUS-CREB, YY1, MAP3K8, NR4A3 and ALK fusions, [7][8][9][10][11][12][13][14][15][16][17][18][19] but most often with no specific clinical or histological features.To better characterize these rearranged proliferations, our group carried out a clinicopathological and further molecular analysis of 41 cases previously identified as harboring a fusion in comparison with 160 control cases without fusion, all collected from the RENAPE and NETMESO/MESOPATH networks.

Population
The cohort consisted of 41 patients treated for rearranged mesotheliomas/neoplasms; the control group included 160 patients treated for neoplasms without rearrangement, the slides for whom were reviewed by the expert pathologists of the Rare Peritoneal Tumors network (RENAPE), and/or the Pleural Mesothelioma and Other Rare Pleural Tumors network (NETMESO/MESOPATH), according to the standardized procedure of certification.One sample was used for each patient.Tumors with clinical annotations were retrieved from the French MESOBANK and its clinicopathological database.Clinical data recorded were age at the time of diagnosis, sex, size, site and number of the lesions, treatment by surgery and/or chemotherapy, duration of follow-up, and survival.The histopathological criteria analyzed were architecture, cellular size and shape, nuclear grade and shape, necrosis, infiltration of the underlying structures, and tumor microenvironment.

Governance
Tissue samples from the French MESOBANK were collected in agreement with all applicable laws, rules, and requests of French and European authorities.These samples were prepared by Centre de Ressources Biologiques of Leon Berard Cancer Center [BB-0033-00050, Lyon France, whose activity is declared to the Ministry of Research (DC-2008-99 and AC-2019-3426)].This study was approved by the review board of the MESOBANK (number 2021-11-17-MesoFusion) and is compliant with General Data Protection Regulation requirements and the French national data protection commission (Commission nationale de l'informatique et des libertés, CNIL; number R201-004-241 on 31 March 2022).

Histological examination
All slides [including hematoxylin, phloxine, and saffron stain, and immunohistochemistry (IHC) staining] were reviewed by two independent pathologists (NB and SL) and classified according to the 2021 WHO classification. 20

Immunohistochemistry
A three-step IHC was carried out on 4 mm-thick formalinfixed paraffin-embedded (FFPE) tissue sections on a BenchMark Ventana autostainer in the biopathology department of the Leon Berard Cancer Center.The antibodies used were raised against pan-cytokeratins, CK5/6, calretinin, WT1, BAP1, CEA, BerEP4, ERa, and PAX8.The proliferation index was assessed by Ki67 nuclear staining (MiB1 clone).The source and dilutions of the antibodies are listed in Supplementary Table S1, available at https://doi.org/10.1016/j.esmoop.2024.103644.The tumor immunophenotype was considered as classical of mesothelial differentiation when calretinin, CK5/6, and/or WT1 were found positive, and BerEp4/CEA negative.Conversely, a doubtful profile of mesothelial differentiation was considered when calretinin, CK5/6, and/or WT1 were found negative, and BerEp4/CEA negative or focally positive.

Whole-exome capture RNA sequencing
Exome-based RNA capture sequencing was carried out on FFPE samples for both rearranged and non-rearranged cases, as part of the MESOPATH/RENAPE diagnostic procedure, and retrieved retrospectively from our files.We targeted mesothelial proliferations with unusual clinical or pathological features.The data were analyzed to detect fusion genes and small nucleotide variations and to compare expression profiles with >6000 other samples using clustering methods.The molecular basis of the technique, the technical protocol, and the bioinformatic algorithms used have been described previously. 21n addition to the detection of gene fusions using the algorithms, the Arriba command-line tool was also used to detect other structural rearrangements with potential clinical relevance.Ward's unsupervised clustering and Uniform Manifold Approximation and Projection (UMAP) were carried out in the R software (v4.2.2) environment, using the cluster and UMAP packages, respectively, on all cases of the cohort (N ¼ 41, Supplementary Table S2, available at https://doi.org/10.1016/j.esmoop.2024.103644)for comparison with the transcriptomic data of other tumors retrieved retrospectively from our files, including angiomatoid fibrous histiocytoma (AFH, N ¼ 18), solid and papillary mesothelial tumor (N ¼ 12), pleural and peritoneal EM (N ¼ 51, and N ¼ 37, respectively), and SM (N ¼ 40, and N ¼ 2, respectively).

Statistical analysis
The statistical analysis was carried out on the rearranged cases.Clinical and pathological data were compared using univariate analyses.Survival was investigated using the KaplaneMeier method, log-rank test, and Cox regression analysis.The statistical software R with package RcmdrPlugin.EZR 22 was used.A P value of <0.05 was considered significant.
ALK-rearranged EM (N [ 7).This group was composed exclusively of females (7/7, 100%), and the mean age was significantly lower than in the other groups [27.8 years (range: 11-37 years), P value: <0.0001].Tumors were multinodular and histologically they presented as extensive and diffuse papillary proliferations of mesothelial cells (Figure 2A) lining the peritoneal surface or superficially infiltrating the peritoneum.Notably, the mesothelial cells around the papillary axes, when clearly visible, appear cuboidal or cylindrical with a homogeneous appearance (Figure 2B).The papillae were edematous, focally infiltrated by foamy cells or psammoma (Figure 2C and D).Hyaline deposits were variably present within the stroma.Infiltration of the papillae was absent or superficial.Nuclear atypia was rare, there was approximately 1 mitosis per 2 mm 2 , and no necrosis.Tumor cells expressed a classical immunophenotype of mesothelial differentiation, with no BAP1 loss.Nuclear PAX8 staining was always present and Ki67 was <5% (Figure 2E-H).Four were treated with a combination of cisplatin or carboplatin and pemetrexed, and one by an anaplastic lymphoma kinase (ALK) inhibitor (alectinib) before surgery.Five patients were treated by CRS þ HIPEC.Two of them were treated with carboplatin and paclitaxel or with a programmed cell death protein 1 (PD-L1) inhibitor (durvalumab) as adjuvant therapy.Among those without HIPEC, one underwent neoadjuvant chemotherapy with a combination of cisplatin and pemetrexed, followed by CRS only.The last patient underwent regular surveillance without surgical or chemotherapeutical intervention (Table 1).This group had a prolonged overall survival; there was no death nor recurrence over a mean follow-up of 56.7 months (range: 8-149 months).[ 10).These patients were predominantly female (6/10, 60%), and the mean age was 56.7 years (range: 30-89 years).Half (5/10, 50%) of the cases arose from the peritoneum, 30% (3/10) from the tunica vaginalis testis, and 20% (2/10) from the pleura.The disease presentation was characterized by a multinodular spread, except for one case that was localized.Two histological patterns were observed according to the site of the proliferations.In the peritoneum/tunica vaginalis testis, tumors closely resembled its ALK-rearranged counterpart, displaying extensive and diffuse papillary proliferation of mesothelial cells (Figure 2I).A distinguishing feature was the fringe-like cellular distribution around the papillae (Figure 2J), setting it apart from the typical pattern observed in ALK fusion cases.The papillae contained a myxoid stroma associated with sometimes foamy cells and/ or psammoma focally (Figure 2K) as well.Variable hyaline deposits were present within the stroma.Infiltrative propensity was either absent or superficial.All tumors were low grade, without necrosis or severe atypia, and there was approximately 1 mitosis per 2 mm 2 .IHC revealed a classical profile of mesothelial differentiation with no loss of BAP1.PAX8 was almost consistently expressed (6/7, 85.7%), with a Ki67 proliferation index of <5% (Table 1, Figure 2L-O).The two pleural cases appeared more aggressive, with marked atypia and a diffuse sheet-like fashion infiltration of the pleural tissue (Figure 2P-R), which was sometimes associated with a dense chronic inflammation at the periphery of the tumor.The nuclear grade was severe and the mean mitotic count was 5 per 2 mm 2 .No foamy cells, psammoma bodies, or necrosis were recognized.The tumors had a doubtful profile of mesothelial differentiation and no BAP1 loss.PAX8 was consistently expressed (2/2, 100%), with a Ki67 proliferation index of 40% (Table 1, Figure 2S-V).For peritoneal cases, the patients were treated by a combination of cisplatin and pemetrexed followed by a CRS for one case (1/5, 20%), by a CRS for one case (1/5, 20%), and with CRS þ HIPEC for two cases (2/5, 40%); one was not treated due to his age (1/5, 20%).The cases of tunica vaginalis testis were treated by surgical resection (3/3, 100%; Table 1).This group of peritoneal and vaginal sites had an intermediate overall survival; there was no death over a mean follow-up of 38 months (range: 3-86 months) and one recurrence at 58 months.In contrast, the two pleural cases had an extremely unfavorable outcome, patients succumbing to their illness in the month following diagnosis.

MAP3K8-rearranged EM (N
EWSR1/FUS::ATF1-rearranged epithelioid tumors (N [ 8).These patients were predominantly female (5/8, 62.5%), and the mean age was 40 years (range: 6-87 years).More than half of the cases arose from the peritoneum (5/8, 62.5%), and the others from the pleura.A multinodular spread was observed in 6/8 cases (75%).In six cases, wellcircumscribed neoplasms enclosed within a thick fibrous capsule, often accompanied by dense lymphoid cuffing were observed (Figure 3A and B).The predominant architectural pattern was solid (Figure 3C), although macrocysts and microcysts (Figure 3D) were discernible, with the smaller cysts containing serous fluid (Figure 3E).In two cases, the tumor was composed of epithelioid cells arranged in a sheet-like pattern or forming small tubular structures surrounding serous fluid.At higher magnification, the epithelioid cells exhibited ill-defined borders, eosinophilic cytoplasm, relatively round but slightly irregular nuclear membranes, vesicular chromatin with prominent nucleoli, and rare mitotic figures.The tumors had a doubtful profile of mesothelial differentiation, with a low or absent calretinin expression.BAP1 loss was observed in 1 case (1/ 8, 12.5%).PAX8 expression was focal in only 1 case (1/8, 12.5%).The Ki67 proliferation index was <5% in 5/6 cases, and in 1 case this ranged from 10% to 20% (Figure 3F-K).For peritoneal cases, 3 patients were treated by CRS þ HIPEC (3/5, 60%), and 1 of them received a combination of cisplatin and pemetrexed as adjuvant treatment (1/5, 20%).The remaining 2 patients were treated by CRS only without any adjuvant treatment (2/5, 40%; Table 1).This group displayed intermediate overall survival; there were two deaths and two instances of recurrence recorded.The mean follow-up was 16.2 months (range: 8-40 months).For pleural cases, only one patient succumbed to their illness 18 months after diagnosis, and two recurrences were reported at 10 and 65.3 months after diagnosis.Two cases were treated with conventional chemotherapy, with or without external radiotherapy, while the third was treated with tocilizumab followed by pleuropneumonectomy and HIPEC, and after surgery, continued with tocilizumab.Conversely, pleural cases exhibited better overall survival; the mean follow-up was 32.1 months (range: 13-65.3months).
EWSR1::YY1-rearranged EM (N [ 8).Patients were predominantly female (5/8, 62.5%), and the mean age was 65.8 years (range: 51-84 years).Tumors were found predominantly in the peritoneum (7/8, 87.5%).The disease presentation was characterized in half of the cases by a multinodular spread (4/8, 50%).Microscopic examination revealed, at low magnification, small tumor nodules scattered throughout, sometimes accompanied by prominent lymphoid aggregates (Figure 3L).The predominant histological pattern consisted of epithelioid cells arranged in cords and trabeculae, some with a macrocystic or focally papillary appearance, set against a background of abundant stromal collagen or lightly myxoid stroma (Figure 3M-P).Additionally, three cases exhibited areas with distinct cell arrangements and extensive regions with solid nests and sheets, occasionally displaying pseudocystic structures containing hemorrhagic material or eosinophilic fluid, reminiscent of AFH-like tumors (Figure 3L).Cytologically, tumor cells were large, polygonal, monomorphic epithelioid  cells with well-defined cell borders, centrally located nuclei, with a fine chromatin, and inconspicuous nucleoli.Nuclearto-cytoplasmic ratios varied among the cases.Neither mitotic activity nor tumor necrosis was observed.Immunohistochemical examination generally revealed a classical profile of mesothelial differentiation (6/8, 75%) and a doubtful profile in 25% of cases (2/8, 25%).BAP1 expression was consistently preserved (7/7, 100%) and PAX8 expression was negative (8/8, 100%).The Ki67 was <5% in 5/8 cases; in 3 cases this ranged from 5% to 15% (Figure 3Q-V).All but 1 patient with peritoneal disease were treated by CRS alone or in combination with HIPEC (6/7, 85.7%); the remaining patient was an elderly patient for whom surgery was not considered (1/7, 14.3%).Adjuvant treatment with carboplatin and pemetrexed was proposed for 1 patient (1/ 7, 14.3%; Table 1).This group exhibited intermediate overall survival; one patient died 11 months after diagnosis despite receiving only CRS, and no recurrence was observed during a mean follow-up of 26.8 months (range: 11-69 months).Conversely, in the pleural case, the patient experienced extremely poor survival outcome, succumbing to his illness 8 months after diagnosis.
NR4A3-rearranged EM (N [ 4).These patients were predominantly male (3/4, 75%), and the mean age was 63.8 years (range: 60-68 years).All tumors arose from the peritoneum and were predominantly multinodular and exophytic.Histologically, microcystic architecture was constant, with cysts lined by a single layer of flattened mesothelial cells arranged diffusely in a sieve-like configuration, with limited stroma and eosinophilic secretion (Figure 3W-AA).Notably, there was no cytological atypia, mitotic activity, or necrosis.Additionally, a sharp transition was observed as the microcystic pattern transformed into spindle cell proliferation (within <10% of the tumor surface, Figure 3W).The tumors had a classical profile of mesothelial differentiation.It is worth noting that BAP1 expression remained intact in all cases (4/4, 100%), with no PAX8 expression (4/4, 100%) and low Ki67 <5% (Table 1, Figure 3AB-AD).All patients underwent surgical resection; 2 of them received HIPEC (2/4, 50%), among whom 1 also received a combination of cisplatin and doxorubicin due to a high peritoneal cancer index (peritoneal cancer index ¼ 26, 1/4, 25%).Among the other 2 patients, HIPEC or chemotherapy were not administered due to either treatment refusal or management in a non-expert peritoneal mesothelioma center (2/4, 50%).This group exhibited a prolonged overall survival with no death or recurrence, and the mean follow-up was 60.8 months (range: 14-140 months).Among them, the two patients without adjuvant treatment had a mean follow-up of 101 months (range: 62-140 months).

Clustering analyses
Clustering analysis using gene expression profiling included cases with gene fusions and control cases without gene fusions.The UMAP projection showed that all ALK, NR4A3, and MAP3K8-rearranged EM formed distinct subgroups, with transcriptomic similarities based on the histology and the site of mesothelioma.EWSR1/FUS::ATF1-rearranged epithelioid tumors clustered together, constituting a separate group from AFH.Similarly, EWSR1::YY1-rearranged EM all clustered together and stood out from the other neoplasms.SUFU-rearranged SM clustered with some epithelioid/sarcomatoid pleural mesotheliomas (Figure 4).They were also distinct from the sarcomas and carcinomas of the Leon Berard Cancer Center transcriptomic database (data not shown) providing strong evidence of their mesothelial lineage.No mutation was identified, except for two pleural MAP3K8-rearranged cases with TP53 mutation, as well as three SUFU-rearranged cases with either BAP1 mutation or PI3KCA/BAP1 co-mutation.Additionally, the analysis of papillary and solid mesothelial tumors harbored a divergent transcriptomic profile.

DISCUSSION
We have expanded herein the spectrum of rearranged mesothelial tumors and defined novel epithelioid (mesothelial) proliferations with distinct histological growth patterns associated with oncological courses.We found a female predominance, except for cases harboring NR4A3   -E).Mesothelial differentiation was doubtful (F-I).BAP1 was retained, PAX8 was negative, and the Ki67 proliferation index ranged from <5% to 10%-20% (J and K).ESWSR1::YY1-rearranged cases (L-V) displayed scattered tumor nodules and epithelioid cells arranged in cords and trabeculae (L-P).Some cases resembled angiomatoid fibrous histiocytoma-like tumors (L).Immunohistochemically, 75% exhibited classical mesothelial profile, while 25% had a doubtful profile (Q-T).BAP1 was retained, PAX8 was negative, and Ki67 proliferation ranged between <10% and 15%-30% (U and V).NR4A3rearranged cases (W-AD) displayed a constant microcystic architecture (W-AA) without atypia, mitotic activity, or necrosis.The transition into spindle cell proliferation occupied <10% of the tumor surface (W).These cases showed a classical mesothelial differentiation profile (AB and AC).BAP1 was retained, PAX8 was negative, and the Ki67 proliferation index was <5% (AD).SUFU-rearranged cases (AF-AL) exhibited a uniform appearance with hypo-and hypercellular areas (AF-AG), occasionally displaying a chevron-like pattern.Spindle-shaped cells arranged in a bland fashion within a fibrous or myxoid stroma were moderately atypical (AH and AI).BAP1 was retained, PAX8 was positive in one case, and the Ki67 index ranged from 40% to 60% (AJ-AL).
and SUFU malignant mesothelioma, and most patients were around 60 years at diagnosis, but those harboring ALK or EWSR1/FUS::ATF1 gene fusions were younger.When located in the peritoneum, ALK and MAP3K8-rearranged EM were characterized by a typical papillary growth pattern, often superficial.They were frequently associated with foamy cells and/or psammoma in peritoneal and vaginal testicular sites, with MAP3K8-rearranged mesothelioma often displaying fringe-like cellular distribution of the cylindrical tumor cells around the papillae, as seen in a case report. 13They all expressed a classical profile of mesothelial differentiation and a PAX8 expression.They presented as lowgrade malignancies and patients had a very long overall survival.In contrast, MAP3K8-rearranged pleural proliferations harboring TP53 mutation appeared as poorly differentiated tumors.The overall survival was pejorative; patients succumbed to their illness within 1 month of diagnosis.
NR4A3-rearranged tumors have been recently described in a series of five cases and well characterized histologically. 18This subset of tumors, both herein and in the recently published paper, presents as a unique microcytic lesion closely resembling adenomatoid tumors, but there was no mutation of TRAF7 or CDC42 which is characteristic of the latter. 23WSR1::YY1 and EWSR1/FUS::ATF1-rearranged lesions represent emerging entities, and the debate about their nature is still ongoing.7,[9][10][11][12][14][15][16]24 The EWSR1::YY1 and EWSR1/FUS::ATF1-rearranged tumors reported herein typically presented as cystic nodules that have AFH-like features.Although EWSR1::ATF1 and FUS::ATF1 fusions are reported in clear-cell sarcomas and AFH, 25,26 the cases herein with EWSR1/FUS::ATF1 fusions were very close to the AFH cluster, but clearly separated on the UMAP projection.The other mesotheliomas and EWSR1::YY1-rearranged lesions were more distant from the AFH cluster.This disagrees with what was reported by the Antonescu group who found a loose clustering of conventional mesotheliomas, EWSR1::YY1 fused lesions (N ¼ 2), and EWSR1/ FUS::ATF1 fused lesions (N ¼ 3).12 Their study was based on their methylation profiling and included a large number of mesotheliomas (N ¼ 79) retrieved from the Heidelberg mesothelioma study (GSE164269), and from The Cancer Genome Atlas (50 colorectal adenocarcinoma, 50 lung adenocarcinoma, and 73 mesotheliomas). Their analysis as   discriminant enough to separate those mesothelial lesions from the other malignant tumors, but not sufficient to decipher the spectrum of those rare rearranged mesothelial tumors.12 We demonstrated herein that EWSR1/FUS::ATF1 fused tumors should probably be considered as distinct mesothelial tumors.This is crucial because these tumors often exhibit an incomplete mesothelial immunophenotype, with no or little calretinin, and variable CK5-6 and WT1 expressions, as previously reported.[7][8][9][10]12,14,15,24 Lastly, for the group harboring SUFU gene fusion, the histological appearance is deceptively reassuring, with monotonous spindle-shaped cells within a homogeneous fibrous or myxoid stroma.As the immunohistochemical profile was inconclusive for mesothelial proliferation, except for BAP1 loss, the diagnosis of mesothelioma was established mainly based on their transcriptomic profile.Interestingly these tumors form a distinct cluster with pleural sarcomatoid mesotheliomas and exhibit a short overall survival.Of note, SUFU as GLI1-3 and SMO are involved in the hedgehog signaling pathway which plays a crucial role in regulating normal cell growth and differentiation. When his pathway is not properly controlled, however, it can lead to abnormal hedgehog signaling, resulting in the development and progression of various aggressive human cancers.This includes neoplastic transformation, tumor progression, metastasis, and drug resistance.Abnormal activation of the hedgehog pathway has been associated with various cancers.As SUFU negatively regulates the hedgehog pathway, 27 targeting the abnormal activity of SUFU holds promise as a therapeutic strategy to impact GLI1, a pivotal contributor to the advancement of multiple cancer types via the hedgehog signaling pathway.It is important to note, however, that as of now, this approach has shown promise in cellular and animal models [28][29][30] and a few cases of clinically established mesotheliomas, due to the low frequency of the hedgehog signaling pathway alterations.31

Conclusion
This study significantly broadens the spectrum of mesothelial tumors associated with fusions, offering insight into novel epithelioid (mesothelial) proliferations with distinctive histological appearances, molecular profiles, and prognoses to guide adapted treatments for patients.

Figure 1 .
Figure 1.Presentation of cohort and analysis of overall survival of cohort.(A) Presentation of cohort.(B-E) Analysis of overall survival of cohort.The univariate analysis found that being female (P value: 0.0346, B), having a peritoneum or vaginal testicular (VT) site (P value: <0.001, C), harboring ALK, MAP3K8, or NR4A3rearranged fusions (P value: 0.016, D), or exhibiting a Ki67 <10% (P value: <0.001, E) were associated with longer overall survival.

Figure 2 .
Figure2.Histological patterns and immunophenotype of mesothelioma rearranged-ALK or MAP3K8.ALK-rearranged cases (A-H) displayed extensive papillary proliferation of mesothelial cells, featuring hyaline or edematous papillae infiltrated by small-sized psammoma bodies and foamy cells.The mesothelial cells around the papillary axes appeared cuboidal or cylindrical with a consistent appearance (A-D).These cases exhibited classical mesothelial differentiation (E and F), conserving BAP1 and PAX8þ expression with a Ki67 proliferation index <5% (G-H).MAP3K8-rearranged peritoneal/vaginal testicular (VT) cases (I-O) resembled the ALK counterpart but showed a fringe-like cellular distribution around the papillae and a myxoid stroma within the papillae, distinguishing them (I-K).They also exhibited classical mesothelial differentiation (L and M), conserving BAP1 and PAX8þ expression, and a Ki67 proliferation index of <5% (N and O).MAP3K8-rearranged pleural cases (P-V) showed marked atypia, diffuse infiltration, and a doubtful mesothelial differentiation profile (S and T), yet maintained BAP1 and PAX8 expression with a Ki67 proliferation index of 40% (U and V).

Table 1 .
Clinical characteristics and immunohistochemical features of the cohort (N [ 41)